Curcumin induced photodynamic therapy mediated suppression of quorum sensing pathway of Pseudomonas aeruginosa: An approach to inhibit biofilm in vitro

doi:10.1016/j.pdpdt.2019.101645

Photodiagnosis and Photodynamic Therapy

Volume 30, June 2020, 101645

https://doi.org/10.1016/j.pdpdt.2019.101645 Get rights and content

Highlights

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Curcumin mediated APDT shows better bacteriostatic than bactericidal activity.
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The thickness of biofilms was reduced from >30 μm to <5 μm after APDT.
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Singlet oxygen is primarily responsible for cytotoxic reactions caused by APDT.
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The inhibition was due the downregulation of quorum sensing mechanism.
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This in turns wipes out EPS that helps in biofilm formation.

Abstract

Objective

The objective of this study was to inhibit the Pseudomonas aeruginosa biofilm through curcumin-mediated antimicrobial photodynamic therapy (A-PDT).

Background

The mechanism behind A-PDT mediated photoinactivation depend upon reactive oxygen species (ROS) production, like singlet oxygen and free radicals.

Methods

To evaluate the antibacterial efficacy of curcumin induced A-PDT on P. aeruginosa by colony forming unit (CFU) while antibiofilm action was determined by the use of crystal violet, XTT, congored binding assay and confocal laser scanning microscope (CLSM).

Results

We found that curcumin with 10 J/cm² of light reduces P. aeruginosa biofilm more efficiently than without light. Extracellular polymeric substances (EPS) production was also reduced by approx 94 % with 10 J/cm² of light dose. CLSM images showed that the thickness of biofilms were reduced from >30 μm to <5 μm after treatment with curcumin followed by 10 J/cm² of light irradiation. Curcumin showed better bacteriostatic activity than bactericidal activity. Singlet oxygen is primarily responsible for photodamage and cytotoxic reactions caused by curcumin-mediated APDT. Genes involved in quorum sensing (QS) pathway was also found to be inhibited after APDT. Curcumin with 5 J/cm² light inhibits QS genes and on increasing light dose i.e10 J/cm², we found a drastic reduction in gene expression.

Conclusion

We conclude that the curcumin mediated A-PDT inhibits biofilm formation ofP. aeruginosa through QS pathway by the action of singlet oxygen generation which in turn reduced EPS of the biofilm.

Introduction

Pseudomonas aeruginosa is an opportunistic human pathogen, it can cause acute and a chronic infection such as urinary tract infections, burn infections, pulmonary infection as well as many other diseases especially in an immune compromised patient [1]. P. aeruginosa has emerged as multidrug resistance (MDR) that leads to serious health problem especially in developing countries [2]. These MDR strains are related with increased mortality that leads to long hospitalization process sometimes ended up with a surgery costing too much [3]. P. aeruginosa has the capacity to form biofilm in many environment that make antibiotic treatments inefficient and leads to chronic infectious biofilm-based diseases [4]. Biofilm is a 3-dimensional structured microbial community that is embedded in a self-produced extracellular polysaccharides matrix (EPS). Bacterial adherence to the surface in order to form biofilm leads to the changes in the phenotype. This is due to the changes in metabolic pathways which enhanced resistance against antibacterial compounds and leads to increase pathogens virulence [5]. These changes are control mechanisms and are regulated by signaling molecules, quorum sensing signaling principle is best studied on P. aeruginosa in the case of biofilm formation [7]. Quorum sensing (QS), a communication system dependent on cell density plays an essential role in controlling virulence factors, cell adhesion antimicrobial resistance and biofilm formation [8,9]. Currently, P.aeruginosa has four known interconnected QS communication systems:las, rhl, Pseudomonas quinolone (pgs), and iqs. Las and rhl are N-acylatedhomoserine lactone (acyl-HSL)-mediated signaling systems. The las system consists of an auto-inducer (lasI) synthesizing the N-3-oxo-dodecanoyl-homoserinelactone auto-inducer (3OC12-HSL). The rhl system, on the other hand, produces N-butanoyl-homo serine lactone (C4-HSL), an auto-inducer synthesized by an auto-inducer rhlI [10]. These signals stimulate exoenzyme production and regulation of virulence factorhence leads to microbial adherence to surface for biofilm formation [9]. Biofilms are resistant to antibiotic therapy and immune protectant, due to the emergence of resistant against large number of antibiotics has put a burden to mankind. Biofilm reduces the penetration of many antibiotics particularly groups of aminoglycosides that leads to resistance.The mechanism of such resistance was due to the overproduction of exopolysaccharide alginate, reduced metabolic activity as well as wrong use of antibiotics treatment which leads to the emergence of highly resistant isolates of bacteria that reduces the efficiency of antibiotics [11]. These infections become very dangerous with diminishing possibility of antibiotic uses, this is clear that there is an urgent need of alternative antimicrobial treatment methods for chronic diseases that are resistant to conventional antibiotic treatment [12]. Antimicrobial photodynamic therapy (A-PDT) as a non-antibiotic therapy is suggested as an alternative approach to inhibit pathogenic microorganisms. A-PDT is a technique used to treat local infections that has a potential to kill a wide range of microorganisms such as resistant bacteria, viruses, fungi, protozoa, and yeasts, [12]. It takes less time than conventional antibiotic therapy, which was also confirmed by our result. Only a few minutes are sufficient to kill many fold of a bacterial cell. A-PDT is defined as a medical treatment that contains a combination of visible light and the non-toxic photoactivable dye or photosensitizer (PS) in the presence of oxygen. When PS absorb photons of an appropriate wavelength of light leads to the formation of cytotoxic reactive oxygen species (ROS) that causes cell death [13]. A-PDT is divided into two types of mechanism, free radicals generation such as hydroxyl (HO·) i.e Type I and singlet oxygen (¹O₂) production i.e Type II. TypeI and TypeII may occur at the same time depending on the PS and its micro environment. We have used a natural compound that has quorum sensing inhibitor QSI activity [9]. Curcumin or diferuloylmethane [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] is a major and natural product extracted from turmeric roots that have a variety of functions and it is traditional indian medicine. It has pharmacological effects in a wide range involves antioxidants, anti-inflammatory, anti-microbial and anti-carcinogenic [14]. Curcumin absorbs blue light within the visible spectrum (300−500 nm). Blue light has low tissue penetration due to scattering and bimolecular absorption, so the use of curcumin in A-PDT is limited to topical use for superficial wounds (e.g. skin and mouth) [15]. In this article, we have attempted to determine the mechanism behind A-PDT mediated photoinactivation of P. aeruginosaby using curcumin as a PS with irradiation of visible region blue light (405 nm wavelength). We also want to explore the effect of A-PDT on biofilm formation.

Section snippets

Bacterial strain

Pseudomonas aeruginosa MTCC 3541 (Microbial Technology Institute, Chandigarh, India) has been used in this study. The microorganism was subculture in Luria Bertani (LB) broth (Himedia labs, Mumbai, India) then incubated at 37 °C.

Photosensitizer and light

Curcumin solution (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione,Himedia, India) was prepared and dissolved in dimethyl sulphoxide (DMSO) of higher concentration after that working solution was prepared in sterile distilled water and stored in the dark

Planktonic cell reduction after APDT

Antimicrobial photoinactivation of P. aeruginosa by curcumin was evaluated by the plate counting method. Curcumin alone at 6.75 mM concentration showed a reduction of 2.12 log₁₀ whereas when treated with 5 J/cm² of light dose it showed 4.26 log₁₀ reduction in cell count while 4.62 log₁₀ reductions were shown when we increase light doses to 10 J/cm² (Fig. 1). There was no reduction in the exclusively light treated group.

Intercellular ROS generation

The intracellular ROS was not enhanced by curcumin with or without light

Discussion

Antimicrobial resistance has become a global problem and a major threat to the well-being of the community. As a result of resistance, conventional antibiotics have become ineffective. Due to the appearance of multidrug resistance in bacteria and their accumulation as a biofilm, the situation becomes worsened [25]. Biofilm is an assembly of sessile bacteria that persist embedded in the self-produced extracellular polymeric substances (EPS) [26]. It provides stability of biofilm structure and

Conclusion

We conclude that curcumin induced A-PDT inhibits the biofilm formation more efficiently through ROS generation specifically Type II photochemistry. The mechanism behind inhibition of biofilm was suppression of the QS system in P. aeruginosa which inturns wipe out EPS. We observed that curcumin in combination with light inhibited QS systemextensively.This study further revealed that 405 nm light played a key role in inhibiting the biofilm formation through QS system. Hence, we proposed curcumin

Acknowledgment

The authors would like to acknowledge DST grant SR/NM/NS-41/2016(G), university sophisticated instruments facility (USIF), AMU. LM was supported by CSIR-RA09/112(0615)2k19EMR-Z.

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¹: Both authors contributed equally.

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Curcumin induced photodynamic therapy mediated suppression of quorum sensing pathway of Pseudomonas aeruginosa: An approach to inhibit biofilm in vitro

Highlights

Abstract

Objective

Background

Methods

Results

Conclusion

Introduction

Section snippets

Bacterial strain

Photosensitizer and light

Planktonic cell reduction after APDT

Intercellular ROS generation

Discussion

Conclusion

Acknowledgment

Braz. J. Infect. Dis.

Photodiagnosis Photodyn. Ther.

Int. J. Antimicrob. Agents

Gene.

Photodiagnosis Photodyn. Ther.

Photochemphotobiol B.

J. Glob. Antimicrob. Resist.

Interaction between extracellular lipase LipA and the polysaccharide alginate of Pseudomonas aeruginosa

BMC Microbiol.

Epidemiology and characteristics of metallo-β-lactamase-producing Pseudomonas aeruginosa

Infect. Chemother.

The role of bacterial biofilms in chronic infections

Apmis

Natural conjugative plasmids induce bacterial biofilm development

Nature

Inhibiting N-acyl-homoserine lactone synthesis and quenching Pseudomonas quinolone quorum sensing to attenuate virulence

Front. Microbiol.

Sodium ascorbate as a quorum sensing inhibitor of P seudomonas aeruginosa

J. Appl. Microbiol.

The hierarchy quorum sensing network in Pseudomonas aeruginosa

Protein Cell